Imaging systems and methods using fluorescent nanodiamonds

ABSTRACT

Imaging systems and methods using fluorescent nanodiamonds are disclosed. The imaging systems and methods including applying a time-varying magnetic field to a specimen containing fluorescent nanodiamonds and comparing the fluorescence obtained with different magnetic fields to provide an image of the specimen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage of International Application No.PCT/US2016/026791, filed Apr. 8, 2016, which claims priority to U.S.Provisional Patent Application No. 62/145,466, filed Apr. 9, 2015, eachof which is incorporated herein by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

The present subject matter was made with U.S. government support. TheU.S. government has certain rights in this subject matter. This work wassupported through grant number HL006087-02 BBC from the National Heart,Lung, and Blood Institute, National Institutes of Health.

BACKGROUND

Autofluorescence from naturally-occurring fluorescent biomolecules andfixative agents make it difficult to separate useful fluorescence fromunwanted fluorescence due to overlapping emission spectra. Therefore,autofluorescence limits the capabilities of tissue and animal imaging.Even sophisticated spectral un mixing techniques cannot always reliablyand accurately separate useful signal from background fluorescence.

BRIEF SUMMARY

One aspect of the invention provides an imaging method including: (a)acquiring a first fluorescent image of an object of interest impregnatedwith fluorescent nanodiamonds; (b) applying a magnetic field to thefluorescent nanodiamonds in order to decrease fluorescence of thefluorescent nanodiamonds; (c) acquiring a second fluorescent image ofthe object of interest; and (d) subtracting the second fluorescent imagefrom the first fluorescent image to produce a resulting image.

This aspect can have a variety of embodiments. The object of interestcan be a biological target. The biological target can be selected fromthe group consisting of: a cell, a plurality of cells, a tissue, anorgan, and an organism.

The magnetic field can be generated by a permanent magnet. The magneticfield can be generated by an electromagnet.

The second fluorescent image can be acquired during application of themagnetic field.

Step (d) can be performed on a pixel-by-pixel basis.

The method can further include an additional step (e) of repeating steps(a)-(d) a plurality of times and averaging the resulting images. Theplurality of times can be greater than 10.

Steps (a) and (c) can include applying an absorption wavelength to theobject of interest. Another aspect of the invention provides anon-transitory computer readable medium containing program instructionsexecutable by a processor. The computer readable medium includes: (a)program instructions that acquire a first fluorescent image of an objectof interest impregnated with fluorescent nanodiamonds; (b) programinstructions that apply a magnetic field to the fluorescent nanodiamondsin order to decrease fluorescence of the fluorescent nanodiamonds; (c)program instructions that acquire a second fluorescent image of theobject of interest; and (d) program instructions that subtract thesecond fluorescent image from the first fluorescent image to produce aresulting image.

This aspect can have a variety of embodiments. The second fluorescentimage can be acquired during application of the magnetic field.

Another aspect of the invention provides an imaging method including:(a) applying a time-varying magnetic field to an object of interestimpregnated with fluorescent nanodiamonds to modulate the fluorescenceof the fluorescent nanodiamonds; (b) acquiring a plurality offluorescent images of the object of interest; and (c) for eachcorresponding pixel in the plurality of fluorescent images, calculatinga fluorescence intensity using a lock-in technique.

This aspect can have a variety of embodiments. The object of interestcan be a biological target. The biological target can be selected fromthe group consisting of: a cell, a plurality of cells, a tissue, anorgan, and an organism.

The magnetic field can be generated by a permanent magnet. The magneticfield can be generated by an electromagnet.

Step (b) can include applying an absorption wavelength to the object ofinterest. The plurality of fluorescent images can be acquired by awide-field camera. The plurality of fluorescent images can be acquiredby a confocal microscope.

Another aspect of the invention provides a non-transitory computerreadable medium containing program instructions executable by aprocessor. The computer readable medium includes: (a) programinstructions that apply a time-varying magnetic field to an object ofinterest impregnated with fluorescent nanodiamonds to modulate thefluorescence of the fluorescent nanodiamonds; (b) program instructionsthat acquire a plurality of fluorescent images of the object ofinterest; and (c) program instructions that, for each correspondingpixel in the plurality of fluorescent images, calculate a fluorescenceintensity using a lock-in technique.

Another aspect of the invention provides an imaging method including:(a) applying an absorption wavelength to an object of interestimpregnated with fluorescent nanodiamonds; and (b) after a delay of atleast about 6 nanoseconds, acquiring a fluorescent image of the objectof interest.

This aspect can have a variety of embodiments. The object of interestcan be a biological target. The biological target can be selected fromthe group consisting of: a cell, a plurality of cells, a tissue, anorgan, and an organism.

The delay can be greater than about 10 nanoseconds. The delay can begreater than between about 10 nanoseconds and about 20 nanoseconds.

The absorption wavelength can be generated by a pulsed laser. Theabsorption wavelength can be between about 450 nm and about 650 nm orbetween about 900 nm and about 1300 nm. The absorption wavelength can bebetween about 450 nm and about 650 nm or between about 850 nm and about1350 nm.

Another aspect of the invention provides an imaging method including:(a) applying an absorption wavelength to an object of interestimpregnated with fluorescent nanodiamonds; and (b) after a delay of atleast at least about 4 nanoseconds, acquiring a fluorescent image of theobject of interest.

This aspect can have a variety of embodiments. The object of interestcan be a biological target. The biological target can be selected fromthe group consisting of: a cell, a plurality of cells, a tissue, anorgan, and an organism.

The delay can be greater than about 10 nanoseconds. The delay can begreater than between about 10 nanoseconds and about 20 nanoseconds.

The absorption wavelength can be generated by a pulsed laser. Theabsorption wavelength can be between about 450 nm and about 650 nm orbetween about 900 nm and about 1300 nm. The absorption wavelength can bebetween about 450 nm and about 650 nm or between about 850 nm and about1350 nm.

Another aspect of the invention provides a non-transitory computerreadable medium containing program instructions executable by aprocessor. The computer readable medium includes: (a) programinstructions that apply an absorption wavelength to an object ofinterest impregnated with fluorescent nanodiamonds; and (b) programinstructions that, after a delay of at least at least about 6nanoseconds, acquire a fluorescent image of the object of interest.

In certain aspects, imaging systems are disclosed, comprising: animaging stage for mounting a specimen; a radiation source configured toexcite the triplet excited state of fluorescent nanodiamonds anddirected on specimen; a fluorescence detector configured to detecttriplet-triplet fluorescent nanodiamond fluorescence and configured todetect fluorescence from the specimen; and an apparatus configured toapply a time-varying magnetic field to the specimen.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the nature and desired objects of thepresent invention, reference is made to the following detaileddescription taken in conjunction with the accompanying drawing figureswherein like reference characters denote corresponding parts throughoutthe following views.

FIG. 1 depicts the absorption (excitation) and emission spectrums ofdiamond nitrogen-vacancy centers.

FIG. 2 depicts an imaging method according to an embodiment of thepresent subject matter.

FIG. 3 depicts an example of pixel-by-pixel subtraction of a secondfluorescent image from a first fluorescent image according to anembodiment of present subject matter.

FIG. 4 depicts an imaging method according to another embodiment of thepresent subject matter.

FIG. 5 depicts an imaging method according to another embodiment of thepresent subject matter.

FIG. 6 depicts an imaging system according to an embodiment of thepresent subject matter.

FIG. 7A depicts the energy level diagram of negatively-charged nitrogenvacancy (NV) centers. FIG. 7B depicts a field of view containing FNDs.FIG. 7C depicts the intensity modulation of the FND depicted in FIG. 7Aupon application of a modulating magnetic field with 0.1 Hz frequencyand 100 Gauss amplitude.

FIG. 8A is a scanning confocal image of FNDs on the surface of a slide.FIG. 8B is a scanning confocal linage taken under identical imagingconditions of the same field of view after the addition of ˜1 μM ALEXAFLUOR® 647 dye, which has a comparable emission spectrum to that of theFNDs. The fluorescence from the high concentration of ALEXA FLUOR® 647dye completely obscures the fluorescence of the FNDs. FIG. 8C is abackground-free linage of the same field of view after processing imagesin the presence of the high ALEXA FLUOR® 647 dye background (as in FIG.8B). The difference between pairs of images collected with and withoutthe magnetic field was computed and 1000 of these difference images wereaveraged together to generate the processed image. Through thisprocessing, images of the diamonds shown in FIG. 8A are recovered fromlinages with high background like FIG. 8B.

FIG. 9A is a frame of a movie taken by a scanning confocal microscope.FIG. 9B shows the wide field background-free image after processing themovie pixel-by-pixel using a lock-in algorithm. FIG. 9C depicts theapplication of the lock-in algorithm to a bright pixel (horizontalcoordinate, x=109 and vertical coordinate, y=111 from the top leftcorner of the image in FIG. 9A) corresponding to a FND. FIG. 9Dillustrates the same lock-in algorithm applied to a dark pixel(horizontal coordinate, x=137 and vertical coordinate, y=107 from thetop left corner of the image in FIG. 9A) corresponding to thebackground.

FIG. 10A depicts the fluorescence image acquired with conventionalspectral unmixing techniques of the forequarters of a mouse afterinjection of FNDs. The image is an overlay of the background channel(top left) and the FND channel (top right). The site of injection of theFNDs is visible in the composite but the lymph node cannot bedistinguished. FIG. 10B shows the same mouse imaged with the pairwiseimage subtraction technique. The lymph node is clearly visible in theprocessed image (top right) that was produced by pairwise subtraction offluorescence images (top left) with the magnetic field on and off. FIG.10C depicts the same images as FIG. 10B obtained with the wide-fieldlock-in technique. FIG. 10D depicts an image of the opened mouse chestcavity obtained with pairwise subtraction background free detection. Thelymph node and the point of initial injection (white arrows) are clearlyvisible. FIG. 10B depicts the same image as FIG. 10D obtained withwide-field lock in background free detection. FIG. 10F depicts theintensity as a function of time for points in the images where there areFNDs (1 and 3) and two background spots (2 and 4). The location of thefour points are indicated on the images in FIGS. 10B and 10D.

FIG. 11A is an image of a lymph node (LN) injected with silica-coatedFNDs without applied magnetic field. FIG. 11B is an image of the samefield of view during application of a magnetic field of ˜100 Gauss. FIG.11C depicts the sum of 20 subtracted images of FIG. 11B from FIG. 11A.The FNDs are observed as bright spots in the otherwise dark field. FIGS.11D and 11E depict the sums of 20 such subtractions when magnetic fieldwas always OFF and ON, respectively.

FIG. 12A is a two-photon FLIM image of a different region of the same LNused in FIGS. 11A-11E. Longer lifetimes (indicated by red color) arebelieved to be due to FNDs, for which the reported lifetimes range from˜10-20 ns. FIG. 12B is a background-free image of the same field of viewobtained by subtracting images without and with a magnetic field, andadding 10 such subtractions.

FIG. 13 shows an imaging system including an alternating magnetic fieldapparatus and a total internal reflection fluorescence microscopeaccording to certain embodiments.

FIG. 14 is a schematic showing the general components of a modulatedmagnetic field fluorescence imaging apparatus.

FIG. 15 shows various configurations for applying a modulated magneticfield according to certain embodiments.

FIG. 16 is a schematic showing elements of a small animal imaginginstrument according to certain embodiments.

FIG. 17 is a schematic showing elements of a small animal imaginginstrument modified for magnetic field modulation according to certainembodiments.

FIG. 18 is a schematic showing elements of a small animal imaginginstrument for magnetic field modulation according to certainembodiments.

FIG. 19 shows a modulated magnetic field fluorescence imaging apparatusaccording to certain embodiments.

FIG. 20 shows a modulated magnetic field fluorescence imaging apparatusaccording to certain embodiments.

FIG. 21 shows a modulated magnetic field fluorescence imaging apparatusaccording to certain embodiments.

FIG. 22 is a schematic showing elements of a hand held modulatedmagnetic field fluorescence imaging apparatus according to certainembodiments.

DEFINITIONS

The present subject matter is most clearly understood with reference tothe following definitions:

As used herein, the singular form “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. “About” canbe understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

As used herein, the term “background-free image” refers to an imagehaving a sufficiently high signal-to-noise ratio (SNR) such that thefluorescence of fluorescent nanodiamonds can be distinguished from thefluorescence of background elements such as endogenous proteins.Suitable SNRs include those greater than about 1:1, about 1.5:1, about2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1,about 9:1, and about 10:1.

As used herein, the terms “comprises,” “comprising,” “containing,”“having,” and the like can have the meaning ascribed to them in U.S.patent law and can mean “includes,” “including,” and the like.

As used herein, the term “fluorescent image” refers to an imagerepresenting one or more fluorescent emissions. For example, thefluorescent emissions can be between about 625 nm and about 825 nm.Fluorescent images are typically obtained typically obtained by applyingan absorption wavelength to an object of interest and simultaneously orafter a delay, capturing an image of fluorescent emissions. Fluorescentimages can be obtained using a variety of devices including fluorescentmicroscopes. The fluorescent image can, in some embodiments, be atwo-dimensional image consisting of a plurality of pixels, each of whichcan be a numerical representation of the intensity of fluorescence as atparticular location.

As used herein, the term “fluorescent nanodiamond” (abbreviated as“FND”) refers to nanodiamonds that exhibit fluorescence when exposed toan appropriate absorption (excitation) spectrum. This fluorescence canbe caused by the presence of nitrogen-vacancy (NV) centers, where anitrogen atom is located next to a vacancy in the nanodiamond. Theabsorption (excitation) and emission spectrums of silica-coated FNDshaving a diameter of about 100 nm are depicted in FIG. 1. The absorption(excitation) spectrum generally lies between about 450 nm and about 600nm with an excitation peak at about 565 nm. (FNDs can also be excited bya two-photon process in the wavelength region between about 900 nm andabout 1300 nm.) The emission spectrum generally lies between about 625nm and about 750 nm, with an emission peak at about 700 nm. (In theworking examples described herein, the samples were excited at 575 nmand a PTI fluorometer was used to obtain the excitation spectra.)

As used herein, the term “nanodiamond” refers to diamonds having alargest dimension of less than about 100 um. For example, the largestdimension of a nanodiamond can be less than: about 100 nm, about 90 nm,about 80 nm, about 70 nm, about 60 nm, about 50 nm, about 40 nm, about30 nm, about 20 nm, about 20 nm, about 10 nm, and the like.

As used herein, the term “object of interest” refers to any object ofwhich a fluorescent image is desired. An object of interest can be abiological target, for example, a living organism or a sample of anorganism. The object of interest can be an organism, one or more organs,one or more tissues, and/or one or more cells. Although the examplesdescribed herein are largely directed toward biological imaging, FNDsand the methods described herein can be used to increase the single tonoise in any imaging application in which a modulated magnetic field canbe applied to the sample. For example, FNDs and the methods describedherein can be used to image and study flow or morphology in a highbackground environment.

Unless specifically stated or obvious from context, the term “or,” asused herein, is understood to be inclusive.

By “specifically binds” is meant recognition and binding to a target(e.g., polypeptide, cell, surface antigen, and the like), but which doesnot substantially recognize and bind other molecules in a sample, forexample, a biological sample.

The term “subject” as used herein, refers to any organism that issuitable for being imaged by the methods described herein. Suchorganisms include, but are not limited to, human, dog, cat, horse, cow,sheep, goat, mouse, rat, guinea pig, monkey, avian, reptiles, bacteria,fungi, viruses, and the like.

The term “tissue” as used herein, refers to a subject's body.Nonlimiting examples of tissues include tissues from organs such asbrain, heart, lung, liver, stomach, pancreas, colon, rectum, intestines,blood vessels, arteries, and the like.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (aswell as fractions thereof unless the context clearly dictatesotherwise).

DETAILED DESCRIPTION

Nitrogen vacancy centers in nanodiamonds are unique fluorescent sourcesthat do not photobleach or blink. Remarkably, the fluorescence intensityof these fluorescent nanodiamonds can be modulated by a magnetic fieldof moderate strength (˜0.01T). Furthermore the fluorescence lifetime ofnanodiamonds (˜10-20 ns) is longer than the lifetime (≤5-6 ns) of mostfluorophores that contribute to auto fluorescence. Both of theseproperties of nanodiamonds can be used to achieve background-freeimaging. The modulation of the fluorescence intensity with appliedmagnetic field is a unique feature of the fluorescent nanodiamonds,which in combination with their other features enables a number of novelimaging applications. Background-free imaging is one of theseapplications.

The discovery allows background-free imaging of fluorescent nanodiamondsin tissue samples and in vivo, where conventional imaging is difficultdue to background fluorescence. We present several techniques to reduceor eliminate background florescence by exploiting properties of thefluorescent nanodiamonds. In particular, magnetic field modulation ofthe fluorescence intensity offers a simple, robust, and easily adaptablemethod to obtain background-free imaging in a variety of imagingmodalities, i.e., fluorescence microscopy, confocal fluorescencemicroscopy, and wide-field fluorescence animal imaging.

In one embodiment of the present subject matter, subtracting an imageacquired with the magnetic field from one without the field collectedunder otherwise identical conditions eliminates constant backgroundfluorescence while highlighting the diamond fluorescence that isspecifically reduced in one image.

In another embodiment, the field is modulated sinusoidally while imagesare acquired. Phase-sensitive detection of the modulated intensity canthen be achieved by postprocessing for camera-based imaging or throughlock-in techniques in confocal-based imaging. This technique could beadapted for use in wide-field and confocal imaging systems. Importantly,this technique makes use of conventional continuous wave illumination.

Yet another embodiment makes use of the long excited state lifetime ofthe fluorescent nanodiamonds to reject shorter-lived backgroundfluorescence. This technique relies on a pulsed laser and time-gated orlifetime imaging. Fluorescent nanodiamonds can be imaged with two-photonapproaches, facilitating these lifetime-based background rejectiontechniques.

Referring now to FIG. 2, an imaging method 200 is provided.

In step S202, a first fluorescent image is acquired of an object ofinterest impregnated with fluorescent nanodiamonds. The fluorescentimage can be acquired using conventional fluorescent imaging devices asdescribed herein.

In step S204, a magnetic field is applied to the fluorescentnanodiamonds in order to decrease fluorescence of the fluorescentnanodiamonds. The magnetic field can be applied, for example, with apermanent magnet or an electromagnet. The magnetic field modulates thefluorescence intensity of the fluorescent nanodiamonds such that thefluorescence intensity of the fluorescent nanodiamonds is less than inthe first fluorescent image.

In step S206, a second fluorescent image is acquired of the object ofinterest. Preferably, the second fluorescent image has identicalparameters to the first fluorescent image other than the decreasedfluorescence intensity of fluorescent nanodiamonds as a result of theapplication of the magnetic field. The second fluorescent image isacquired while the magnetic field is applied.

In step S208, the second fluorescent image is subtracted from the firstfluorescent image to produce a resulting image. The subtraction can beperformed on a pixel-by-pixel basis. For example, as shown conceptuallyin FIG. 3, a second 10×10 pixel fluorescent image can be subtracted froma first 10×10 pixel fluorescent image to produce a resulting image.Because the only difference between the first fluorescent image and thesecond fluorescent image is the modulation of fluorescent nanodiamonds,the background fluorescence (e.g., from endogenous proteins) will becanceled out by the subtraction to produce a background-free fluorescentimage.

Pixel-by-pixel subtraction can be performed manually or can beautomated. A variety of commercially-available computer programs canperform image subtraction including, for example, MATLAB® softwareavailable from The MathWorks, Inc. of Natick, Mass. and IMAGEJ softwareavailable from the National Institutes of Health athttp://rsbweb.nih.gov/ij/.

In step S210, steps S202-S208 are repeated a plurality of times (e.g.,greater than 10) and the resulting images are averaged on apixel-by-pixel basis to improve the imaging quality.

Referring now to FIG. 4, another imaging method 400 is provided.

In step S402, time-varying magnetic field is applied to an object ofinterest impregnated with fluorescent nanodiamonds to modulate thefluorescence of the fluorescent nanodiamonds. The time-varying magneticfield can vary cyclically. For example, the magnitude of the magneticfield can vary sinusoidally. The magnetic field can be varied bymodulating the current applied to an electromagnet or by modulating thedistance between the magnet (either a permanent magnet or anelectromagnet) and the object of interest.

In step S404, a plurality of fluorescent images of the object ofinterest are acquired. Preferably, the plurality of fluorescent imagesare captured at a plurality of points within a cycle. The sampling canoccur at regular or irregular intervals and can, but need not, bematched to the frequency of the phase-modified magnetic field. Theplurality of fluorescent images can be acquired pixel-by-pixel using apoint imager such as a confocal microscope or multiple pixels at a timeusing a wide-field imager.

In step S406, the fluorescence intensity of each pixel is calculatedusing a lock-in technique. Lock-in techniques multiply the fluorescenceintensity in each pixel of the plurality of fluorescent images by theamplitude of the magnetic field at the time of each respectivefluorescent image and then calculate the intensity of the appropriatelyfiltered or processed resulting products. All background fluorescencewill not fluctuate in phase with the magnetic field modulation over allimages and will therefore average to zero. The fluorescence intensity ofthe fluorescent nanodiamonds will vary in phase with the magneticintensity and, therefore, average to half of the amplitude of themagnetic intensity.

Lock-in techniques are described in publications such as RichardBurdett, “Amplitude Modulated Signals—The Lock-in Amplifier,” inHandbook of Measuring System Design (2005) and Stanford ResearchSystems, Inc., “About Lock-In Amplifiers: Application Note #3,”available athttp://www.thinksrs.com/downloads/PDFs/ApplicationNotes/AboutLIAs.pdf.Generally speaking, single-pixel inputs obtained from point detectorscan processed directly by conventional lock-in amplifiers available fromsuppliers such as Stanford Research Systems, Inc. of Sunnyvale, Calif.,while algorithms can be written using software such as MATLAB® to stepthrough each set of corresponding pixels in a series of fluorescentimages and perform a lock-in technique to produce a resultingbackground-free image.

If the fluorescent images are acquired pixel-by-pixel, a lock-intechnique can be applied for that particular pixel before acquiringanother pixel. If the images are acquired using a wide-field imager, alock-in technique can be applied on a pixel-by-pixel basis after allimages are acquired.

Referring now to FIG. 5, another imaging method 500 is provided.

In step S502, an absorption wavelength (e.g., a brief pulse of 5 ns orless) is applied to an object of interest impregnated with fluorescentnanodiamonds. This absorption wavelength will excite FNDs, but may alsoexcite various background elements such as endogenous proteins.

In step S504, after a delay of at least at least about 6 nanoseconds,acquiring a fluorescent image of the object of interest. After 6nanoseconds, most (if not all) background fluorescence will havedissipated, while the FNDs continue to emit photons. Thus, abackground-free image can be captured without the need for the imageprocessing algorithms described above.

Referring now to FIG. 6, the methods described herein can be implementedin hardware and/or software. For example, FIG. 6 depicts a system 600including a fluorescent imaging device 602 such as a fluorescentmicroscope and a computer 604. The fluorescent imaging device 602 caninclude an excitation light source, an image sensor (e.g., including acharge-coupled device (CCD), complementary metal-oxide-semiconductor(CMOS) chip, photomultiplier tube (PMT), or avalanche photodiode (APD)),one or more lenses, and a filter adapted to block undesired wavelengths.The computer 604 can be a special-purpose or general-purpose computercan be communicatively coupled with the fluorescent imaging device 602via communication standards such as parallel or serial ports, UniversalSerial Bus (USB), USB 2.0, Firewire, Ethernet, Gigabit Ethernet, and thelike.

As understood by those of skill in the art, computer 604 can includevarious components such as a display device, a processor, and/or astorage device.

Display device can be any device capable of displaying graphics and/ortext. Examples of display devices include a cathode ray tube (CRT), aplasma display, a liquid crystal display (LCD), an organiclight-emitting diode display (OLEO), a light-emitting diode (LED)display, an electroluminescent display (ELD), a surface-conductionelectron-emitter display (SED), a field emission display (FED), anano-emissive display (NED), an electrophoretic display, a bichromalball display, an interferometric modulator display, a bistable nematicliquid crystal display, and the like.

Processor is an electronic device (also known as a central processingunit or microprocessor) capable of executing instructions stored ashardware and/or software. Suitable processors are available frommanufacturers such as Intel Corporation of Santa Clara, Calif. orAdvanced Micro Devices (AMD) of Sunnyvale, Calif.

Storage device can include persistent storage devices such as magneticmedia (e.g. tapes, disks), optical media (e.g. CD-ROM, CD-R, CD-RW, DVD,HD DVD, BLU-RAY DISK®, Laserdisk), punch cards, and the like. Storagedevice can also include temporary storage devices known as memory (e.g.,random access memory).

In some embodiments, motion correction is applied to compensate formotion in the camera and/or the subject. For example, motion correctioncan be applied to a time series of images to correct for in-plane motionat the sub-pixel level.

In one embodiment, a non-rigid deformation map is calculated pairwisebetween each image and a common reference image using an optical flowmethod, which iteratively maximizes the local cross-correlation imagesubsets at different resolutions. The initial time frame can be chosenas a common reference for both images series acquired with externalfield ON and OFF in order so that the ON and OFF images areco-registered. A subpixel spline based interpolation can be used forapplication of the non-rigid deformation to minimize loss of spatialresolution. Following motion correction, the time series of images canbe averaged to reduce random fluctuation due to noise thereby creatingan average image for the ON and OFF. A difference image between ON andOFF motion corrected averages can be used to analyze the contrast of theprobe signal by subtraction of the background tissue. This signal fromthe nanodiamond can then be overlaid on the starting image.

As described herein, the present invention relates to use of fluorescentnanodiamonds to image an object of interest. Fluorescent nanodiamondssuitable for use in the present invention and methods for making suchfluorescent molecules are well-known in the art. see, e.g.,Nanodiamonds: Applications in Biology and Nanoscale Medicine (D. Ho ed.,Springer 2009); Molecular Imaging (R. Weissleder et al. eds., 2010);Medical Nanotechnology and Nanomedicine (Perspectives in Nanotechnology)(H. F. Tibbals ed., CRC Press 2010); Molecular Fluorescence (B. Valeured., Wiley-VCH 2012); Introduction to Nanomedicine andNanobioengineering (Wiley Series in Biomedical Engineering andMulti-Disciplinary Integrated Systems) (P. N. Prasad ed., Wiley 2012);Yu et al., J. Am. Chem. Soc. 127:17604-17605 (2005); Mochalin et al.,Nature Nanotechnology 7:11-23 (2012); Epstein et al., Nature Physics1:94-98 (2005); Chow et al., Sci. Transl. Med. 3:73ra21 (2011);Awschalom et al., Sci. Am. 297:84-91 (2007); and Wilson, Phys. Today64:17-18 (2011).

The fluorescent nanodiamonds can be provided as a solution, emulsion,suspension, microsphere, particle, microparticle, nanoparticle,liposomes, and the like.

In aspects of the invention, the fluorescent nanodiamonds are directlycontacted with a sample (e.g., when impregnating the fluorescentnanodiamond in a sample in vitro, ex vivo, in situ, etc.). For example,fluorescent nanodiamonds can be impregnated in a sample by injection(e.g., microinjection) or use of a delivery vehicle. Suitable deliveryvehicles are well-known in the art. Nonlimiting examples include lipidvesicles or other polymer carrier materials, lipoplexes (see, e.g., U.S.Patent Application Publication No. 2003/0203865; and Zhang et al., J.Control Release, 100:165-180 (2004)), pH-sensitive lipoplexes (see,e.g., U.S. Patent Application Publication No. 2002/0192275),reversibly-masked lipoplexes (see, e.g. U.S. Patent ApplicationPublication Nos. 2003/0180950), cationic lipid-based compositions (see,e.g., U.S. Pat. No. 6,756,054; and U.S. Patent Application PublicationNo. 2005/0234232), cationic liposomes (see, e.g., U.S. PatentApplication Publication Nos. 2003/0229040, 2002/0160038, and2002/0012998; U.S. Pat. No. 5,908,635; and International Publication No.WO 01/72283), anionic liposomes (see, e.g., U.S. Patent ApplicationPublication No. 2003/0026831), pH-sensitive liposomes (see, e.g., U.S.Patent Application Publication No. 2002/0192274; and AustralianPublication No. 2003/210303), antibody-coated liposomes (see, e.g., U.S.Patent Application Publication No. 2003/0108597; and InternationalPublication No. WO 00/50008), cell-type-specific liposomes (see, e.g.,U.S. Patent Application Publication No. 2003/0198664), liposomescontaining nucleic acid and peptides (see, e.g., U.S. Pat. No.6,207,456), liposomes containing lipids derivatized with releasablehydrophilic polymers (see, e.g., U.S. Patent Application Publication No.2003/0031704), lipid-entrapped molecules (see, e.g., InternationalPublication Nos. WO 03/057190 and WO 03/059322), lipid-encapsulatedmolecules (see, e.g., U.S. Patent Application Publication No.2003/0129221; and U.S. Pat. No. 5,756,122), other liposomal compositions(see, e.g., U.S. Patent Application Publication Nos. 2003/0035829 and2003/0072794; and U.S. Pat. No. 6,200,599), stabilized mixtures ofliposomes and emulsions (see, e.g., European Publication No. EP 1 304160), emulsion compositions (see, e.g., U.S. Pat. No. 6,747,014), andmicro-emulsions (see, e.g., U.S. Patent Application Publication No.2005/0037086).

In some aspects of the invention, the fluorescent nanodiamonds areadministered in vivo to a subject (e.g., delivering the fluorescentnanodiamond to a target object within the subject, including, but notlimited to, a target cell, tissue, organ, organism, infectious agent, avims, a bacteria, fungus, a parasite, and the like). When administeredto a subject, the fluorescent nanodiamonds can be provided. as apharmaceutical composition including a pharmaceutically acceptablecarrier. The term “pharmaceutically acceptable” means approved by aregulatory agency or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the therapeutic is administered. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil, olive oil, gel (e.g., hydrogel),and the like. Saline is an exemplary carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions.

Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol, and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. Oral formulation caninclude standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, and the like. Examples of suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin, the contents of which are hereby incorporated by reference inits entirety.

In embodiments, the imaging agent may be administered through differentroutes, including, but not limited to, oral, parenteral, buccal andsublingual, rectal, aerosol, nasal, intramuscular, subcutaneous,intradermal, and topical. The term parenteral as used herein includes,for example, intraocular, subcutaneous, intrapelitoneal, intracutaneous,intravenous, intramuscular, intraarticular, intraarterial,intrasynovial, intrastemal, intrathecal, intralesional, and intracranialinjection, or other infusion techniques. One of ordinary skill in theart would readily understand that the formulation should suit the modeof administration.

Formulations suitable for administration include aqueous and non-aqueoussterile solutions, which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation isotonic with theblood of the intended recipient, and aqueous and non-aqueous sterilesuspensions which may include suspending agents and thickening agents.The formulations may be presented in unit-dose or multi-dose containers,for example, sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, immediately prior to use.Extemporaneous solutions and suspensions may be prepared from sterilepowders, granules and tablets commonly used by one of ordinary skill inthe art.

For oral administration in the form of a tablet or capsule, thefluorescent nanodiamonds can be combined with an oral, non-toxic,pharmaceutically acceptable, inert carrier such as lactose, starch,sucrose, glucose, methyl cellulose, magnesium stearate, dicalciumphosphate, calcium sulfate, mannitol, sorbitol and the like. For oraladministration in liquid form, the fluorescent nanodiamonds can becombined with any oral, non-toxic, pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water, and the like. Moreover, whendesired or necessary, suitable binders, lubricants, disintegratingagents, and coloring agents can also be incorporated into the mixture.Suitable binders include starch, gelatin, natural sugars such as glucoseor β-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth, or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes, and the like. Lubricants used in thesedosage forms include sodium oleate, sodium stearate, magnesium stearate,sodium benzoate, sodium acetate, sodium chloride, and the like.Disintegrators include, without limitation, starch, methyl cellulose,agar, bentonite, xanthan gum, and the like.

The fluorescent nanodiamonds used in the methods of the presentinvention can also be administered in the form of liposome deliverysystems. Such delivery systems are well known in the art, and includebut are not limited to, unilamellar vesicles, large unilamallarvesicles, and multilamellar vesicles. Liposomes can be formed from avariety of phospholipids, such as cholesterol, stearylamine, orphosphatidylcholines.

The administration of the fluorescent nanodiamonds to a subject can beby a general or local administration route. For example, the fluorescentnanodiamonds may be administered to the subject such that it isdelivered throughout the body. Alternatively, the fluorescentnanodiamonds can be administered to a specific organ or tissue ofinterest.

The fluorescent nanodiamonds should have sufficient emission to assurereliable diagnosis. The amount of fluorescent nanodiamonds to becontacted with a sample or introduced into a subject in order to providefor detection can readily be determined by those skilled in the art. Forexample, increasing amounts of the fluorescent nanodiamonds can beapplied or given to a subject until the fluorescent nanodiamonds aredetected by the detection method of choice. In addition, those skilledin the art are also familiar with determining the amount of timesufficient for the fluorescent nanodiamonds to become associated with atarget object. The amount of time necessary can easily be determined byintroducing a detectable amount of the fluorescent nanodiamonds into asubject and then detecting the fluorescent nanodiamonds at various timesafter administration.

In some aspects, the fluorescent nanodiamonds can be associated with amolecule that preferentially binds to the target object. Such moleculesare well-known in the rut, and include but are not limited totarget-binding agents having one or more target recognition moieties forthe selective binding of the target-binding agents (and fluorescentnanodiamond) to a target molecule. The target recognition moiety isconfigured to specifically bind to a target molecule of a particularcell, tissue, organ, receptor, surface antigen, organism/infectiousagent, and the like.

Examples of target recognition moieties include, but are not limited to,an antigen, ligand, receptor, one member of a specific binding pair,polyamide, peptide, carbohydrate, oligosaccharide, polysaccharide, lowdensity lipoprotein (LDL) or an apoprotein of LDL, steroid, steroidderivative, hormone, hormone-mimic, lectin, drug, antibiotic, aptamer,DNA, RNA, lipid, an antibody, an antibody-related polypeptide, and thelike. In embodiments, the targeting moieties are polypeptides,carbohydrates, or lipids. The targeting moieties can also be antibodies,antibody fragments, or nanobodies. In other embodiments, the targetrecognition moiety can be a molecule or a macromolecular structure(e.g., a liposome, a micelle, a lipid vesicle, or the like) thatpreferentially associates or binds to a particular tissue, receptor,organism/infectious agent, and the like.

One of ordinary skill in the art would readily understand how to makethe fluorescent nanodiamond conjugates contemplated herein. For example,the fluorescent nanodiamonds can be covalently or non-covalentlyassociated with the target binding agent/moiety. See Vaijayanthimalal etal., Nanomedicine 4:47-55 (2009); Vaijayanthimalal et al., Biomaterials33: 7794-7802 (2012); Hartmann et al., Chemistry—A European Journal18:21, 6485-6492 (2012); Mochalin et al., Nat. Nanotechnology 7:11-23(2012); Weng et al., Diamond and Related Materials 22:96-104 (2012);Alhaddad et al., Small 7:3087-3095 (2011); Krueger, J Mater. Chem.21:12571-12578 (2011); and Liu et al., Nanoscale Research Letters5:1045-1050 (2010); Rurack, Supramolecular Chemistry Meets Hybrid(Nano)Materials: A Brief LookAhead, pages 689-700 of The SupramolecularChemistry of Organic-Inorganic Hybrid Materials (Wiley, 2010).

In some embodiments, the FNDs are silica-coated FNDs. Methods forcreating silica-coated nanodiamonds are described in A. Bumb et al.,“Silica encapsulation of fluorescent nanodiamonds for colloidalstability and facile surface functionalization,” 135 Journal of theAmerican Chemical Society 7815-18 (2013).

Working Example #1—Magnetic Modulation of FND Emission

FIG. 7A depicts the energy level diagram of NV centers in diamondshowing spin-triplet (m_(s)=0 and m_(s)=±1) ground and excited states aswell as the singlet metastable state. NV⁻ centers can be opticallyexcited over a broad range of wavelengths (450-650 nm) (green arrows).NV⁻ centers in the m_(s)=±1 sublevels of the excited states have ahigher probability to decay via the metastable state (grey dashedarrows) than to the m_(s)=±1 sublevels of the ground state. From themetastable state, NV centers predominantly transition to the m_(s)=0sublevel of the ground state without emitting visible light. Therefore,in the absence of a magnetic field, NV⁻ centers are rapidly pumped intothe m_(s)=0 sublevel of the ground state when excited. This results inan initial increase in fluorescence emission intensity as steady stateis reached. In the presence of a magnetic field, the m_(s)=0 andm_(s)=±1 states are mixed, making the decay pathway through themetastable singlet state accessible and therefore decreasing thefluorescence emission intensity.

FIG. 7A depicts a field of view containing FNDs. FIG. 7B depicts theintensity modulation of the FND depicted in FIG. 7A upon application ofa modulating magnetic field with 0.1 Hz frequency and 100 Gaussamplitude.

To show magnetic modulation of FND emission, a coverslip was preparedwith FNDs. 500 μl of 1 mg/ml poly-L-lysine (PLL) in PBS buffer was mixedwith silica-coated FNDs, deposited on a #1 coverslip, and incubatedovernight. A flow cell was made using double-sided tape to attach thecoverslip to a glass slide.

Movies were obtained using a CARL ZEISS® LSMS LIVE microscope. Eachframe of the movie was a scanning confocal image with 250 ms timeresolution. A 10× ZEISS® objective with 0.3 NA (EC Plan-Neofluar) wasused to introduce the excitation light to stimulate the FNDs and tocollect the FND emission. Samples were excited at 532 nm, emission wasfiltered using a long pass filter LP650 and detected using aphotomultiplier tube (PMT).

An electromagnet (APW Company, Item # EM400-12-212, 4.0″ Diameter RoundElectromagnet) was powered by square wave voltage signal with anamplitude of 0 or 12V and zero offset. The sample was placed ˜13 mm awayfrom the face of the magnet, where the magnetic field strength was ˜100Gauss.

Working Example #2—Background-Free Imaging by Pairwise Subtraction ofFrames with and without Magnetic Field

FIG. 8A is an image of a field of view with ˜40 nm FNDs containing ˜15NV-imaged as in FIG. 7B. FIG. 8B is an image of the same field of viewafter introducing ˜1 μM ALEXA FLUOR® 647 dye solution into the flowcell. This dye has similar emission characteristics as the FNDs so thefluorescence from the FNDs is masked by the background fluorescence ofhigh concentration of the ALEXA FLUOR® 647 dye. FIG. 8C is an image ofthe same field of view after processing images in the presence of thehigh ALEXA FLUOR® 647 dye background (as in FIG. 8B). The differencebetween pairs of images collected with and without the magnetic fieldwas computed and 1,000 of these difference images were averaged togetherto generate the processed image. Through this processing, images of thediamonds shown in FIG. 8A are recovered from images with high backgroundlike FIG. 8B.

Working Example #3—Background-Free Imaging Using Wide-Field Lock-inDetection

FIG. 9A shows a frame of a movie (1000 frames with time resolution 0.25s) taken by a scanning confocal microscope. The details of the imagingare identical to those in FIG. 7B above. A modulating magnetic fieldwith 0.1 Hz frequency and 100 Gauss amplitude was applied during theacquisition of the movie.

FIG. 9B shows the wide field background-free image after processing themovie pixel-by-pixel using a lock-in algorithm.

FIG. 9C depicts the application of the lock-in algorithm to a brightpixel (horizontal coordinate, x=109 and vertical coordinate, y=111 fromthe top left corner of the image in FIG. 9A) corresponding to a FND. Thetop left panel of FIG. 9C illustrates the pixel values as a function oftime. The top right panel of FIG. 9C illustrates the fast Fouriertransform (FFT) as a function of frequency. The middle left panel ofFIG. 9C depicts the pixel values from the top left panel multiplied by areference sine wave 1+sin(2π×0.1×t). The middle right panel of FIG. 9Cdepicts the corresponding FFT. The bottom left panel of FIG. 9C showsthe pixel values multiplied with a reference cosine wave1+cos(2π×0.1×t). The bottom right panel of FIG. 9C depicts thecorresponding FFT.

FIG. 9D illustrates the same lock-in algorithm applied to a dark pixel(horizontal coordinate, x=137 and vertical coordinate, y=107 from thetop left corner of the image in FIG. 9A) corresponding to thebackground.

Referring to both FIGS. 9C and 9D, vertical dashed lines at 0.2 Hz areguides to indicate the values at twice the reference frequency. Means ofthree points around 0.2 Hz in the sine and cosine FFTs were calculatedfor each pixel. The background-free image in FIG. 9B corresponds to thesine and cosine means added in quadrature, i.e, i=sqrt(i cos²+i sin²),where i is the mean intensity and i cos and i sin correspond to themeans calculated from the FFT of the signal multiplied by the cosine andsine functions respectively. Means of the pixel values over 1000 frameswere 173 and 18 for the pixels at (109,111) and (137,107) beforeapplying the lock-in algorithm giving a signal-to-noise ratio of ˜10.Means of the FFT amplitudes for three points around 0.2 Hz are 21.42 and0.28 for the pixels at (109,111) and (137,107) after applying thelock-in algorithm giving a signal-to-noise ratio of ˜77. Thus thelock-in algorithm increased the signal to noise ratio by a factor of ˜8.Computations were implemented in MATLAB® software.

Working Example #4—Background-Free Imaging of Sentinal Lymph Node InVivo Using Wide Field Pairwise Image Subtraction and Lock-in Detection

FIG. 10A depicts an image of the forequarters of a mouse obtained usingconventional spectral unmixing methods to separate the emission of theFNDs (top tight inset, red in overlay) from background fluorescence (topleft inset, white in overlay). FNDs were not detected through the skinin the draining axillary lymph node. FIG. 10B depicts an image of thesame mouse obtained by averaging 475 pairwise-subtracted images with andwithout the magnetic field. The processed image (top right inset, red inoverlay) was overlaid on an unprocessed image obtained with the magneticfield off (top left inset, white in overlay). The white arrows point tothe injection site in the footpad and the location of the auxiliary(sentinel) lymph node. Signal from the FNDs in the lymph node is clearlydetected. FIG. 10C depicts an image obtained by lock-in detection ofemission from FNDs from the same images used to generate FIG. 10B usingthe algorithm described in FIG. 9 (top right inset, red in overlay)overlaid on the unprocessed image obtained with the magnetic field off(top left inset, white in overlay). FIG. 10D depicts an image of thesame mouse's open chest cavity by averaging of images obtained bypairwise subtracting images with and without the magnetic field. Theprocessed image (bottom inset, red in overlay) was overlaid on anunprocessed image obtained with the magnetic field off (top inset, whitein overlay). The white arrows point to the injection site in the frontfootpad and the location of the auxiliary lymph node. FIG. 10E depictsan image obtained from lock-in detection of emission from FNDs from thesame images used to generate FIG. 10D (bottom inset, red in overlay)overlaid on the unprocessed image obtained with the magnetic field off(top inset, white in overlay). In the partially dissected mouse,localization of the FNDs to the lymph node can be clearly seen. FIG. 10Fdepicts the pixel values as a function time corresponding to theselected points in FIG. 10B and FIG. 10D. The pixels selected were over(1) the axillary lymph node and (2) a negative control on the skin inFIG. 10B and (3) the axillary lymph node and (4) a negative control on arib in FIG. 10D. Signal modulation as a result of the applied magneticfield is clearly visible in the lymph node through the skin, as well aswhen the chest cavity was opened. Meanwhile, the skin and rib showedrandom signal as would be expected for the negative control.

Female athymic (nu/nu) mice were purchased from Charles RiverLaboratories at 4-6 weeks of age and housed in a specific pathogen-freeAmerican Association for Laboratory Animal Care approved facility. Allexperiments were approved by the National Cancer Institute's Animal Careand Use Committee. Mice were anesthetized using gas mixtures of 1.5-2.5%isoflurane in O₂ to maintain a respiration rate of ˜30 bpm during theinjection procedure. A volume of 10 μL of an ˜80 mg/mL silica-coatednanodiamond (˜100 nm) solution in PBS (pH7.4) was intradermally injectedinto the front foot pad of each mouse. Previous studies have shown thatthe primary draining LNs from this injection site are the axillary andlateral thoracic LNs.

At 24 hours post-injection, the mice were sacrificed and optical imagingwas performed using a MAESTRO™ CRi spectroscopic optical camera(excitation filter 523 nm, emission filter 675 nm long pass). Imageswere also taken with magnetic modulation in a UVP® BIOSPECTRUM® ImagingSystem equipped with a BIOLITE® MultiSpectral Light Source (excitationfilter 525 nm, emission filter 650 nm long pass) with additionalillumination provided by a green laser (Z-Bolt® # DPSS-100) achieving ˜1mW/cm² intensity. Each mouse was imaged in the instrument on anon-magnetic stage under which the permanent magnet (Catalog # DZ08-N52,K&J Magnetics) could be slid in and out. Images were captured in serieswith magnetic fields off and on and movies stitched together in ImageJsoftware available at http://rsb.info.nih.gov/ij.

FIG. 11A is an image of a lymph node (LN) injected with silica-coatedFNDs without applied magnetic field. FIG. 11B is an linage of the samefield of view during application of a magnetic field of ˜100 Gauss. FIG.11B was then subtracted from FIG. 11A. FIG. 11C depicts the sum of 20such subtracted images. The FNDs are observed as bright spots in theotherwise dark field. FIGS. 11D and 11E depict the sums of 20 suchsubtractions when magnetic field was always OFF and ON, respectively.

Female athymic (nu/nu) mice were purchased from Charles RiverLaboratories of Wilmington, Mass. at 4-6 weeks of age and housed in aspecific pathogen-free American Association for Laboratory Animal Careapproved facility. Animals were used between 6 and 8 weeks of age, andexperiments were approved by the National Cancer Institute's Animal Careand User Committee. Lymphadenectomy of the axillary and lateral thoraciclymph nodes (LN) of the mice was performed. The excised LN was injectedwith silica-coated FNDs. The LN was then fixed in 10% formalin for 3days, washed with PBS, and mounted on a SUPERFROST PLUS® slide (obtainedfrom VWR) using PERMOUNT® medium (obtained from Fisher Scientific) toadhere the coverslip.

Background-free images were obtained by using a Neodymium permanentmagnet (obtained from K&J Magnetics of Jamison, Pa.). A CARL ZEISS® LSMSLIVE microscope was used to obtain the movies. Each frame of the moviewas a scanning confocal image with 1 s time resolution. A 10× ZEISS®objective with 0.3 NA (EC Plan-Neofluar) was used to excite the FNDs andcollect the FND emission. Samples were excited at 532 nm. Emission wasfiltered using a long pass filter (bandpass of 560-675 nm) and detectedusing a PMT.

Working Example #5—Background-Free Imaging Using Fluorescence LifetimeImaging (FLIM)

FIG. 12A is a two-photon FLIM linage of a different region of the sameLN used in FIGS. 11A-11E. Longer lifetimes (indicated by red color) arebelieved to be due to FNDs, for which the reported lifetimes range from˜10-20 ns. FIG. 12B is a background-free image of the same field of viewobtained by subtracting images without and with a magnetic field, andadding 10 such subtractions.

A LEICA® TCS SP5 scanning confocal system was customized to obtain boththe fluorescent lifetime imaging microscopy (FLIM) image (FIG. 12A) andthe background-free confocal image in which a Neodymium permanent magnetwas used to apply the magnetic field (˜100 Gauss) (FIG. 12B). For FIG.12B, 10 subtractions of images with and without magnetic fields wereadded to obtain the final image. An oil immersion objective (LEICA® HCXPL APO CS 40.0× 1.25 NA Oil UV) was used both to excite and to collectthe emission. For the FLIM image, the sample was excited at 930 nm usinga SPECTRA-PHYSICS® MM TAI® laser. For FLIM imaging, emission from theFNDs in the LN was filtered using a band pass filter (BP 607-683 nm) andwas detected using a PICOQUANT™ PICOHARP™ 300 Time-Correlated SinglePhoton Counting (TCSPC) system fitted with single photon avalanche diode(SPAD). For the scanning confocal image (400 Hz scanning speed) of thesame field of view (FOV), the sample was excited at 561 nm and theemission was filtered using band pass filter (600-789 nm) and wasdetected using a PMT.

The present subject matter provides unique, elegant methods that can beeasily incorporated with existing microscope or animal imaging systemswithout coming in contact with the sample. No complicated anderror-prone spectral unmixing is necessary. Fluorescence ofcommonly-used stains and labels cannot be modulated selectively using amagnetic field. Chemical or optical modifications of commonly usedlabels such as GFP and dyes are possible, but these modifications aredifficult to implement, are invasive, and can possibly induce unwantedchanges.

While the present subject matter has been described with reference tothe above embodiments, it will be understood by those skilled in the artthat various changes can be made and equivalents can be substituted forelements thereof without departing from the scope of the subject matter.In addition, many modifications can be made to adapt a particularsituation or material to the teachings of the subject matter withoutdeparting from the essential scope thereof. Therefore, it is intendedthat the subject matter not be limited to the particular embodimentdisclosed as the best mode contemplated for carrying out this subjectmatter, but that the subject matter will include all embodiments fallingwithin the scope of the appended claims.

The functions of several elements may, in alternative embodiments, becarried out by fewer elements, or a single element. Similarly, in someembodiments, any functional element may perform fewer, or different,operations than those described with respect to the illustratedembodiment. Also, functional elements (e.g., modules, databases,computers, and the like) shown as distinct for purposes of illustrationmay be incorporated within other functional elements, separated indifferent hardware or distributed in a particular implementation.

FIG. 13 shows the elements of a modulated magnetic field fluorescenceimaging system. A biological sample of interest is mounted on the AFMstage 11. An apparatus for applying a modulated magnetic field ismounted over the AFM stage. The magnet field applicator is controlled byAFM controller 10 controlled by AFM computer 13. The position of themagnetic field is monitored using XYZ closed-loop scanner 9. A condenserand transilluminator are mounted above the AFM stage and illuminate thebiological sample.

A total internal reflection fluorescence (TIRF) imaging apparatus ismounted below the AFM stage 11. TIRF illumination can be provided by anAr/Kr laser 23 optically coupled to filter 16, telescope 15, opticalfiber 18, and TIRF illuminator 19. Radiation from TIRF illuminator 19 isincident on a beam splitter, which directs the Ar/Kr laser radiationthrough a lens to focus on the biological sample. Ar/Kr laser 23 iscooled with water cooling system 24.

Alternatively, the sample can be irradiated with a xenon-arc lamp suchas a Lambda LS illuminator (Sutter Instrument Co.). Radiation from thexenon arc 25 can be collimated using Epi-IRM illuminator 12, directedonto a beam splitter and focused onto the biological sample.

Fluorescence is collected by the confocal microscope or other suitableimaging system and the fluorescence detected using CCD cameras 14 andvideo camera 22. Imaging optics are controlled and imaging data acquiredusing microscope computer 8.

The modulated magnetic field fluorescence imaging system shown in FIG.13 illustrates the general elements of a suitable imaging system. Ingeneral, the system is configured such that a time-modulated magneticfield is applied to the biological sample. The magnetic field may beapplied to the entire biological sample or a portion of the biologicalsample. The system is configured to irradiate the entire biologicalsample or a portion of the biological sample to cause the biologicalsample to fluorescence. The system is further configured to collect thefluorescence.

In certain embodiments, fluorescent nanodiamonds are used to image abiological sample. A biological sample can be labeled withfunctionalized fluorescent nanodiamonds. The time-varying magnetic fieldis applied at a magnetic strength sufficient to mix the +/−1 and 0triplet states at a high field strength.

FIG. 14 is a schematic summarizing the elements of a modulated magneticfield fluorescence imaging system and includes a modulated magneticfield apparatus, an imaging instrument, a specimen such as a biologicalsample, and a software analysis module. The modulated magnetic field canbe applied using a permanent magnet or an electromagnet. In certainembodiments, a permanent magnet can be moved with respect to thespecimen to provide a time-varying magnetic field. In certainembodiments, an electromagnet is used to apply a time-varying magneticfield with different waveforms.

An imaging instrument referred to in FIG. 14 can include suitablemicroscope imaging optics such as used in confocal microscopy, lightmicroscopy, TIRF microscopy, and FLIM microscopy, or animal imagingsystems such as transillumination scanners or reflectance scanners. Asappropriate, the imaging instrument can include, for example, opticalfilters, lenses, collimators, and beam splitters. An imaging instrumentcan also include devices for detecting fluorescence and opticallyimaging the sample. Optics for irradiating the sample to inducefluorescence and translation stages for moving the magnet, optics,radiation source, and/or imaging components are also included.

The various components can be controlled by software. Image processingcan involve comparison of images obtained within and without a magneticfield applied to the sample or involve comparing the fluorescenceobtained at low and high magnetic field strength.

FIG. 15 shows various configurations of a magnet with respect to asample stage. For example, in certain embodiments an electromagnet canbe mounted above, below and/or to the sides of the sample stage. Anelectromagnet can be used to apply a time-varying magnetic field. Inanother configuration, a permanent magnet can be mounted above, below,and/or to the sides of the sample stage and physically moved toward andaway from the sample stage to change the strength of the magnetic fieldapplied to the sample. As shown in the third scheme, a permanent magnetcan be mounted on an apparatus configured to rotate the magnet about thesample to provide a time-varying magnetic field. In certain embodiments,a permanent magnet can be stationary and a magnetic shield can bedisposed between the magnet and the sample to provide a time-varyingmagnetic field.

FIG. 16 is a schematic showing the general elements of a small animaloptical imaging instruments. The imaging instrument includes a lightsource, a specimen such as a biological sample, a detector, software forcontrolling the light source and the detectors(s) and a user interface.The light source can include multiple excitation filters for controllingthe wavelength range of the excitation irradiation and the detector caninclude multiple filters for selecting the wavelength range of thefluorescence.

FIG. 17 is a schematic showing the general elements of a small animalimaging instrument for obtaining time-modulated magnetic fieldfluorescent images. The instrument shown in FIG. 17 includes a lightsource, specimen such as a biological sample, a detector(s), softwarefor controlling the light source and detector(s), a user interface, anda software interface for controlling a magnetic field applicator forapplying a magnetic field to the specimen. The light source can includemultiple excitation filters for controlling the wavelength range of theexcitation irradiation and the detector can include multiple filters forselecting the wavelength range of the fluorescence. In certainembodiments, the excitation wavelength range and the detectionwavelength range are selected to optimize the contrast of magnetic fieldmodified NV-center nanodiamond fluorescence. The magnetic fieldapplicator can include a permanent magnet or an electromagnet configuredto apply a time-varying magnetic field to the specimen. The softwareaddition can be used to correlate magnetic field-dependent images of thespecimen with optical images of the specimen, and to perform imageprocessing to provide images of the specimen. In the case of imagingbased on magnetic field modified NV-center nanodiamond fluorescenceimages of the specimen or a portion of the specimen obtained with amagnetic field and without a magnetic field or with a lower intensitymagnetic field can be compared reduce or eliminate backgroundfluorescence that does not originate from NV-center fluorescentnanodiamonds.

FIG. 18 shows another embodiment of a small animal imaging instrumentfor magnetic field modulation including a light source, a specimen, adetector, a magnetic field, applicator, software for controlling thelight source, the detector, and the magnetic field applicator, and auser interface. In this embodiment the light source includes a sourceand/or optical system configured to optimize excitation of the NV-centerfluorescent nanodiamond triplet state and a detector and opticsincluding, for example, a filter, selected to optimize detection ofNV-center fluorescent nanodiamond fluorescence.

FIGS. 19-21 show various configurations of a light source, specimen,detector, and magnetic field applicator. FIG. 19 shows a light source,specimen, and detector in a linear configuration with the magnetic fieldapplied from below a rotating stage. FIG. 20 shows the light source,specimen and detector in a confocal configuration in which theexcitation source and the detector(s) are on the same side of thespecimen and with the magnetic field applicator on the opposite side ofthe specimen. FIG. 21 shows an example of imaging instrumentation inwhich detectors are located around the specimen and with a single lightsource an magnetic field applicator. In certain embodiments thedetectors, light source, and magnetic field applicator can be rotatedaround the sample. In certain of the embodiments illustrated in FIG. 21,there can be multiple light sources and magnetic field applicators.

Modulated magnetic field fluorescence imaging apparatus can bestationary systems in which the specimen or biological sample is mountedon a stage and the imaging instrumentation is a standalone system. Incertain embodiments, an imaging apparatus provided by the presentdisclosure can be a portable device such as a hand-held scanner. Asshown, for example, in FIG. 22, a clinical hand-held scanner can includea light source, a magnetic field applicator, a camera or detector,software configured to obtain modulated magnetic field fluorescenceimages of a patient, and an interface between the clinician and thesoftware. In certain embodiments, the elements of the scanner areoptimized to obtain images of magnetic field modified NV-centernanodiamond fluorescence.

In certain embodiments, the fluorescent nanodiamonds are silica coated.Silica-coated nanodiamonds and methods of preparing silica-coatednanodiamonds are disclosed, for example, in WO 2014/014970 A1, which isincorporated by reference in its entirety. Similar methods can be usedto silica coat fluorescent nanodiamonds.

Silica-coated fluorescent nanodiamonds can be functionalized withprobes, affinity molecules, or other entities that can bind to specifictargets of a biological sample. These functionalized silica-coatedfluorescent nanodiamonds can be applied to a biological specimen such asa tissue sample or cells to stain the specimen with the functionalizednanodiamonds. In other embodiments, functionalized nanodiamonds can beadministered in vivo and the magnetically modified fluorescence of thefunctionalized silica-coating nanodiamonds measured through the skin ofa patient. In certain embodiments, following in vivo administration offunctionalized nanodiamonds to a subject, tissue or biological fluid canbe removed from the subject and evaluated for the presence offunctionalized nanodiamonds. Functionalized silica-coated nanodiamondscan be used to probe targets similar to ways in which fluorescent probesare used.

While certain embodiments according to the present subject matter havebeen described, the present subject matter is not limited to just thedescribed embodiments. Various changes and/or modifications can be madeto any of the described embodiments without departing from the spirit orscope of the present subject matter. Also, various combinations ofelements, steps, features, and/or aspects of the described embodimentsare possible and contemplated even if such combinations are notexpressly identified herein.

What is claimed is:
 1. An imaging system comprising: an imaging stagefor mounting a specimen; a radiation source configured to excitefluorescent diamonds and directed on the specimen; a fluorescencedetector configured to detect diamond fluorescence and configured todetect fluorescence from the specimen; and a permanent magnet, whichrotates with respect to the specimen to apply a time-varying magneticfield to the specimen.
 2. The imaging system of claim 1, comprising thespecimen containing the diamonds mounted on the imaging stage.
 3. Theimaging system of claim 1, wherein the radiation source is selected froma group consisting of a laser and a xenon arc lamp.
 4. The imagingsystem of claim 1, wherein the fluorescent diamonds are fluorescentnanodiamonds, and comprise silica-coated fluorescent diamonds orfunctionalized silica-coated fluorescent diamonds.
 5. The imaging systemof claim 1, wherein the fluorescent diamonds are fluorescentnanodiamonds comprise functionalized silica-coated fluorescentnanodiamonds.
 6. The imaging system of claim 1, wherein the permanentmagnet is positioned above, below, or next to the specimen.
 7. Theimaging system of claim 1, wherein the specimen is selected from apatient, an animal, a slide, a tissue, and/or a cell.
 8. The imagingsystem of claim 1, comprising an imaging processing unit for processingan image from data from the fluorescence detector.
 9. The imaging systemof claim 8, wherein the image processing unit is configured to compareimages obtained with a first applied magnetic field provided by thepermanent magnet to the specimen with fluorescence images obtained witha second applied magnetic field provided by the permanent magnet to thespecimen.
 10. The imaging system of claim 9, wherein the first magneticfield and the second magnetic field have a different intensity.
 11. Theimaging system of claim 1, wherein the imaging system is portable. 12.The imaging system of claim 1, wherein the imaging system is configuredto image a portion of the specimen having a dimension from 1 cm to 10cm.
 13. The imaging system of claim 1, wherein the imaging system isconfigured to image a portion of the specimen having a dimension of atleast 1 cm.
 14. The imaging system of claim 1, wherein the imagingsystem is configured to image a portion of the specimen having adimension of less than 0.1 cm.
 15. The imaging system of claim 1,wherein the imaging system is configured to image a portion of thespecimen having a dimension from 0.01 cm to 0.1 cm.
 16. The imagingsystem of claim 1, wherein the radiation source and the fluorescencedetector are configured to move with respect to the specimen.
 17. Theimaging system of claim 1, wherein the radiation source, thefluorescence detector, and the permanent magnet configured to apply atime-varying magnetic field are configured to move with respect to thespecimen.
 18. The imaging system of claim 1, wherein the systemcomprises a plurality of detectors including the fluorescent detector.19. The imaging system of claim 1, wherein the system comprises aplurality of fluorescence detectors arranged around the specimen, theplurality of fluorescence detectors configured to rotate around thespecimen and receive fluorescent signals from the specimen.
 20. Theimaging system of claim 1, wherein the system comprises a plurality offluorescence detectors, and the plurality of fluorescence detectors, theradiation source, and the permanent magnet are arranged around thespecimen, and being configured to rotate around the specimen when thesystem is in operation.
 21. The imaging system of claim 1, wherein thediamonds are nanodiamonds.
 22. The imaging system of claim 1, whereinthe permanent magnet configured to apply a time-varying magnetic fieldto the specimen also applies the time-varying magnetic field to thediamonds at the same time, the diamonds being nanodiamonds.
 23. Theimaging system of claim 1, wherein the fluorescent diamonds arefluorescent nanodiamonds and comprise functionalized fluorescentnanodiamonds.
 24. The imaging system of claim 1, further comprising aplurality of permanent magnets, each of the permanent magnets configuredto apply a time-varying magnetic field to the specimen.